The Department of Veterans Affairs is the largest provider of medical care to people with HIV in the United States; in 2016 ~30,000 Veterans received treatment for HIV from the Veterans Health Administration (VHA). Over the last several decades, the success of antiretroviral therapy (ART) treatment for HIV infection has changed the demographics and phenotype of Veterans living with HIV. Although HIV+ Veterans are living longer, 78% of them are overweight or obese, and they have a two-fold greater risk of developing type 2 diabetes compared to Veterans without HIV. While much of the research on HIV and metabolic disease has focused on the interaction between obesity and the accumulation of ectopic fat in the liver, several recent studies highlight the central role of ectopic fat in skeletal muscle in the pathogenesis of insulin resistance and diabetes. However, this important phenomenon has received little attention in the context of HIV infection and the role of skeletal muscle ectopic fat in the complex interaction between HIV and obesity remains unclear. Defects in adipose tissue lipid storage and regulation are hallmark of both HIV infection and obesity, which leads to a high degree of ectopic fat accumulation in tissues such as the liver and skeletal muscle. While several studies have investigated ectopic liver fat as a risk factor for diabetes in HIV, our novel hypothesis is that impaired glucose tolerance in Veterans with treated HIV and obesity is driven by disproportionately greater ectopic lipid infiltration of skeletal muscle (the primary site of glucose uptake) promoting impaired myocyte bioenergetics and glucose homeostasis (Fig.1). This is supported by our preliminary CT imaging, MRS imaging, and glucose metabolism data that implicate skeletal muscle pathology in HIV associated glucose intolerance: 1) Our CT data show ectopic fat infiltration in muscle is greater in HIV+ diabetics vs nondiabetics; 2) Our MRS data show higher muscle triglyceride content is associated with a slower rate of ATP synthesis; 3) Our metabolic data show lower plasma acylcarnitines (indicating impaired mitochondrial oxidation) correlates with insulin resistance in HIV. In the proposed study, we aim to determine: a) whether the deposition of excess lipid in skeletal muscle in HIV+ Veterans is a phenomenon separate from hepatic fat deposition and a hallmark for T2DM in this population (Aim 1); b) whether skeletal muscle fat accumulates over time in nondiabetic overweight/obese Veterans and is accompanied by reductions in muscle mitochondrial oxidative capacity, ATP production, and muscle force (Aim 2); and c) whether changes in skeletal muscle ectopic fat and mitochondrial oxidative capacity over time are accompanied by reductions in insulin sensitivity and glucose tolerance (Aim 3). To accomplish these aims, we will recruit two cohorts of HIV+ Veterans on long-term ART that will be matched by sex, age, race, BMI and CD4/CD8 ratio. Group 1: 45 Veterans with HIV, obesity and type 2 diabetes; Group 2: 45 Veterans with HIV, obesity and no diabetes. Our study will: 1) determine the contribution of ectopic fat in skeletal muscle vs liver to developing glucose intolerance in HIV; 2) identify temporal changes and determine relationships in skeletal muscle ectopic fat infiltration and impairments in mitochondrial function in HIV; 3) identify temporal changes and clarify relationships between skeletal muscle ectopic fat, liver ectopic fat, mitochondrial function, and glucose tolerance in HIV; 4) establish a foundation for future studies targeting ectopic fat deposition to improve metabolic health; and 5) inform our understanding of lipid pathology and the development of diabetes in treated HIV, yielding opportunities for interventions to advance the healthcare of our Veterans.